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During protein synthesis, the ribosome moves along a single mRNA strand and translates the genetic code into polypeptide sequences. Strong mRNA secondary structures, which have to be unfolded in order to be translated, can slow or even halt protein synthesis. Pseudo-knots (PKs), mRNA secondary structures containing at least two stem-loops, are essential elements in programmed ribosomal frameshifting, for instance, during translation of some viral mRNAs. Here we employ single molecule fluorescence resonance energy transfer (smFRET) to determine reaction rates for specific polypeptide elongation cycles as the ribosome encounters a minimal viral PK structure during mRNA translation. An elongation cycle may be divided into three steps: 1) aminoacyl-tRNA binding to the A-site, 2) translocation of the bound mRNA and tRNAs from the A- and P- sites to the P- and E-sites, and 3) dissociation of deacylated tRNA from the E-site. We use FRET interactions between i) adjacent ribosome-bound tRNAs, ii) aminoacyl-tRNA and ribosomal protein L11 at the A-site, and iii) deacylated tRNA and ribosomal protein L1 at the E-site, to determine the rates of steps 1) - 3) during elongation cycles of ribosomes programmed with mRNAs either containing or lacking a PK. We find that, whereas the presence of a PK has little or no effect on the rates of steps 1) and 2), it strongly decreases (∼2.5-fold) the rate of step 3. Thus, somewhat surprisingly, step 3) appears to be more strongly coupled to the unfolding of PK structure than step 2). In contrast, preliminary results indicate that stem-loop structures can decrease the rates of both steps 2) and 3). Supported by NIH grant R01GM080376.

To kinetically monitor translation-related events at a nucleotide resolution, a series of mRNAs, site-specifically modified at key codon positions with new emissive and responsive isomorphic nucleotides (thC, thU, thG - work withthA is commencing) have been studied. mRNAs modified with thC, thU and thG (excluding thU2 in the start codon) form an initiation complex (70SIC) and show significant spectral changes between the free and 70SIC-bound fluorescent mRNAs. All emissive mRNAs tested containing labeled nucleotides in the second codon facilitate aa-tRNA A-site binding [as part of an aa-tRNA.EF-Tu.GTP ternary complex (TC)] and pretranslocation (PRE) complex formation, although with somewhat different relative efficiencies, and all PRE complexes can be translocated via addition of EF-G.GTP to form postranslocation (POST) complexes. In most cases spectral differences are seen on conversion of 70SIC to PRE complexes and PRE complexes to POST complexes, allowing measurement of the kinetics of such conversions by changes in the fluorescence of labeled mRNAs. Preliminary comparison of tRNA A-site binding kinetics, as monitored by the fluorescent mRNAs, with other measures of PRE complex formation indicate that TC binding to the ribosome is faster than codon-anticodon interaction, which in turn is somewhat faster than tRNA accommodation. These preliminary observations not only show excellent performance of mRNAs modified with our emissive RNA alphabet, but also clearly demonstrate their potential to illuminate the formation and disappearance of discrete intermediates in the polypeptide elongation cycle.

During ribosome-catalyzed polypeptide chain elongation, dissociation of the deacylated tRNA from the E-site has been proposed to be either spontaneous or triggered allosterically by binding of the next cognate ternary complex to the A-site. Using fluorescent labeled tRNAs, we have measured single molecule fluorescence intensities and single molecule FRET between adjacent tRNAs in the ribosome. From these measurements we have been able to determine tRNA occupancy in the ribosome, and thus whether E-site tRNA dissociates before or after A-site occupancy. In the former case there are a maximum of 2 simultaneously bound tRNAs, while the latter case results in transient binding of 3 tRNAs simultaneously. In a total internal reflection florescence microscope, ribosomes were attached to glass microscopes slides via a biotinylated mRNA coding for MRFFRFY.... (single letter amino acid sequence). When synthesis was initiated with tRNAfMet fully charged with formylated-Met, 60-70% of the ribosomes lost their E-site tRNA prior to ternary complex binding (2-tRNA pathway) at the 2nd and 3rd elongation cycles (R and F respectively). In contrast, for synthesis initiated with uncharged initiator tRNAfMet, >90% of the ribosomes followed the 2-tRNA pathway in the 2nd cycle, but only ∼15% in the 3rd cycle (∼75% following the 3-tRNA route, 10% not categorized). In cycles 4 and 5, almost all ribosomes followed the 2-tRNA pathway. Thus, the length of the peptide chain and/or the specific amino acids bound to the P-site tRNA strongly influence the allostery of E-site tRNA dissociation. Such allostery is also sensitive to the presence or absence of polyamines, Mg2+ concentration, and the specific codons in positions 2 and 3. Supported by NIH R01grant GM080376 and NIST ATP grant 70NANB7H7011 through Anima Cell Metrology, Inc.

Metal enhanced fluorescence (MEF), in which a surface plasmon near a noble metal alters the spectral properties of an organic fluorophore, increases fluorescence intensity without a concomitant increase in photobleaching rate. To improve recordings of single molecule fluorescence signals from individual ribosomes, we studied the emission of Cy3- and Cy5-labeled ribosomes and tRNAs attached near 50-80 nm silver colloidal particles on a glass microscope coverslip. The fluorescence of Cy3 and Cy5 labeled initiation complexes (ICs) near 50 nm silver particles was increased 4- and 5-fold, respectively, compared to labeled ICs in the absence of silver colloids. Photobleaching lifetime was not significantly accelerated, resulting in 4-5 fold enhancement of total photon emission before photobleaching. Fluorophores showing enhanced fluorescence were colocalized with the colloidal particles, as detected by light scattering. Other ribosomes or tRNAs that were farther away had intensities similar to those on plain glass. Aggregates of silver colloidal particles themselves produced wavelength-shifted luminescence similar to fluorescence, presumably due to resonance between nearby metal particles. With ribosomes bound to the glass substrate near the silver particles via a short mRNA, interaction between tRNAArg-Cy3 in the ribosomal P-site and fMet-Arg-Phe-tRNAPhe-Cy5 in the A-site had FRET efficiency and dynamics similar to ribosomes farther away and on plain glass. Binding of Cy5-Arg-tRNAArg to ICs labeled with Cy3 on the large subunit protein L11, in the absence of the translocase EF-G, produced FRET efficiency and dynamics characteristic of specific codon-dependent A-site binding. Addition of EF-G reduced FRET efficiency, as expected. These tests demonstrate that the colloidal silver nanoparticles increase fluorescence and total photon emission without compromising the biophysical activity of ribosomes.Supported by NIH grants GM080376 and HG004364.

Transfer-RNA (tRNA) is the key adaptor molecule in protein synthesis, but recent studies demonstrate behavior and interactions that suggest a far greater role in cellular function. This includes priming viral protein synthesis, retrograde nuclear trafficking, binding to cytochrome C, and post-translational arginylation of proteins. Defects in tRNA modifications are also linked to a number of human diseases with relatively unknown mechanisms. Here we describe the use of live-cell imaging techniques to monitor tRNA nuclear-cytoplasmic trafficking and localization in real time via microinjection of fluorescently-labeled tRNA (fl-tRNA) into adherent mammalian cells. These results represent an important step toward our eventual goal of developing assays for the full characterization of the subcellular dynamics of specific isoacceptor tRNAs.

During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule polarized total internal reflection fluorescence (polTIRF) microscopy to characterize tilting and rotational fluctuations within specific domains of EF-G. When EF-G binds to the ribosomal pre-translocation (PRE) complex, domains I and IV of EF-G undergo small rotations (10-15°) in conjunction with translocation, whereas domain III shows a much greater angular change, averaging 50°. Viomycin (Vio), which prevents translocation, reduces the rotational motions of domain III to 10-15° but has virtually no effect on the other domains. Spectinomycin also reduces domain III motions but less strongly than Vio. EF-G binding to ribosomal initiation complexes lacking A-site tRNA gives a similar pattern of domain rotations, but with shorter dwell times. In this case, the large rotation of domain III is barely inhibited by Vio. Irrespective of completion of translocation or presence of A-site tRNA, the initial 10-15° rotations of EF-G domains I, III and IV in the ribosome/EF-G complex indicate that the EF-G initially shifts the minimum of the free energy profile in the direction of translocation, suggesting that EF-G generates a force on the ribosome and/or the mRNA and tRNAs. Near the end of translocation, domain III completes its rotation either to push the mRNA and tRNAs (a working stroke) or to prevent reversal of translocation driven by thermal fluctuations (a ratchet). Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.